Signaling lipids control many of the most important biological pathways, typically by recruiting cognate protein binding targets to cell surfaces, thereby regulating both their function and subcellular localization. A critical family of signaling lipids is that of the phosphatidylinositol polyphosphates (PIPns), which is composed of seven isomers that vary based on phosphorylation pattern. A key protein that is activated upon PIPn binding is Akt, which then plays important roles in regulating the cell cycle, and is thus aberrant in disease. Characterization of protein–PIPn binding interactions is hindered by the complexity of the membrane environment and of the PIPn structures. Herein, we describe two rapid assays of use for characterizing protein–PIPn binding interactions. First, a microplate-based binding assay was devised to characterize the binding of effectors to immobilized synthetic PIPn headgroup–biotin conjugates corresponding to all seven isomers. The assay was implemented for simultaneous analysis of Akt-PH domain, indicating PI(3,4,5)P3 and PI(3,4)P2 as the primary ligands. In addition, density-dependant studies indicated that the amount of ligand immobilized on the surface affected the amplitude of protein binding, but not the affinity, for Akt-PH. Since the PIPn ligand motifs used in this analysis lack the membrane environment and glycerolipid backbone, yet still exhibit high-affinity protein binding, these results narrow down the structural requirements for Akt recognition. Additionally, binding detection was also achieved through microarray analysis via the robotic pin printing of ligands onto glass slides in a miniaturized format. Here, fluorescence-based detection provided sensitive detection of binding using minimal amounts of materials. Due to their high-throughput and versatile attributes, these assays provide invaluable tools for probing and perturbing protein–membrane binding interactions.

We synthesized and characterized a series of zwitterionic, acetate-terminated, quaternized amine diacyl lipids (AQ). These lipids have an inverted headgroup orientation as compared to naturally occurring phosphatidylcholine (PC) lipids; the cationic group is anchored at the membrane interface, while the anionic group extends into the aqueous phase. AQ lipids preferentially interact with highly polarizable anions (ClO4−) over less polarizable ions (Cl−), in accord with the Hofmeister series, as measured by the change in zeta potential of AQ liposomes. Conversely, AQ lipids have a weaker association with calcium than do PC lipids. The transition temperatures (Tm) of the AQ lipids are similar to the Tm observed with phosphatidylethanolamine (PE) lipids of the same chain length. AQ lipids form large lipid sheets after heating and sonication; however, in the presence of cholesterol, (Chol) these lipids form stable liposomes that encapsulate carboxyfluorescein. The AQ:Chol liposomes retain their contents in the presence of serum at 37 °C, and when injected intravenously into mice, their organ biodistribution is similar to that observed with PC:Chol liposomes. AQ lipids demonstrate that modulating the headgroup charge orientation significantly alters the biophysical properties of liposomes. For the drug carrier field, these new materials provide a non-phosphate containing zwitterlipid for the production of lipid vesicles.

Tetraazamacrocyclic complexes of transition metals provide useful units for incorporating multiple coordination interactions into a single protein binding molecule. They can be designed with available sites for protein interactions with donor atom containing amino acid side chains or have labile ligands such as H2O allowing facile exchange. Three configurationally restricted nickel(II) cyclam complexes with either one or two macrocyclic rings were synthesised and their ability to abrogate the CXCR4 chemokine receptor signalling process was assessed (IC50 = 8320, 194 and 14 nM). Analogues were characterised crystallographically to determine the geometric parameters of acetate binding as a model for aspartate. The most active nickel(II) compound was tested in several anti-HIV assays against representative viral strains showing highly potent EC50 values down to 13 nM against CXCR4 using viruses with no observed cellular cytotoxicity (CC50 > 125 μM).

Liposomes consisted of phosphatidylinositol (PI) and phosphatidylcholine (PC) have been utilized as delivery vehicle for drugs and proteins. In the present work, we studied the effect of soy PI on physical properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes such as phase state of lipid bilayer, lipid packing and phase properties using multiple orthogonal biophysical techniques. The 6-dodecanoyl-2-dimethylamino naphthalene (Laurdan) fluorescence studies showed that presence of PI induces the formation of fluid phases in DMPC. Differential scanning calorimetry (DSC), temperature dependent fluorescence anisotropy measurements, and generalized polarization values for Laurdan showed that the presence of as low as 10 mol% of PI induces substantial broadening and shift to lower temperature of phase transition of DMPC. The fluorescence emission intensity of DPH labeled, PI containing DMPC lipid bilayer decreased possibly due to deeper penetration of water molecules in lipid bilayer. In order to further delineate the effect of PI on the physico chemical properties of DMPC is due to either significant hydrophobic mismatch between the acyl chains of the DMPC and that of soy PI or due to the inositol head group, we systematically replaced soy PI with PC species of similar acyl chain composition (DPPC and 18:2 (Cis) PC) or with diacylglycerol (DAG) respectively. The anisotropy of PC membrane containing soy PI showed largest fluidity change compared to other compositions. The data suggests that addition of PI alters structure and dynamics of DMPC bilayer in that it promotes deeper water penetration in the bilayer, induces fluid phase characteristics and causes lipid packing defects that involve its inositol head group.

Electron paramagnetic resonance (EPR) spin-labeling methods make it possible not only to discriminate the cholesterol bilayer domain (CBD) but also to obtain information about the organization and dynamics of cholesterol molecules in the CBD. The abilities of spin-label EPR were demonstrated for Chol/POPC (cholesterol/1-palmitoyl-2-oleoylphosphatidylcholine) membranes, with a Chol/POPC mixing ratio that changed from 0 to 3. Using the saturation-recovery (SR) EPR approach with cholesterol analogue spin labels, ASL and CSL, and oxygen or NiEDDA relaxation agents, it was confirmed that the CBD was present in all membrane suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST). Conventional EPR spectra of ASL and CSL in the CBD were similar to those in the surrounding POPC bilayer (which is saturated with cholesterol), indicating that in both domains, cholesterol exists in the lipid-bilayer-like structures. The behavior of ASL and CSL (and, thus, the behavior of cholesterol molecules) in the CBD was compared with that in the surrounding POPC-cholesterol domain (PCD). In the CBD, ASL and CSL molecules are better ordered than in the surrounding PCD. This difference is small and can be compared to that induced in the surrounding domain by an ~10°C decrease in temperature. Thus, cholesterol molecules are unexpectedly dynamic in the CBD, which should enhance their interaction with the surrounding domain. The polarity of the water/membrane interface of the CBD is significantly greater than that of the surrounding PCD, which significantly enhances penetration of the water-soluble relaxation agent, NiEDDA, into that region. Hydrophobicity measured in the centers of both domains is similar. The oxygen transport parameter (oxygen diffusion-concentration product) measured in the center of the CBD is about ten times smaller than that measured in the center of the surrounding domain. Thus, the CBD can form a significant barrier to oxygen transport. The results presented here point out similarities between the organization and dynamics of cholesterol molecules in the CBD and in the surrounding PCD, as well as significant differences between CBDs and cholesterol crystals.

Conjugated linoleic acids (CLA) are found naturally in dairy products. Two isomers of CLA, that differ only in the location of cis and trans double bonds, are found to have distinct and different biological effects. The cis 9 trans 11 (C9T11) isomer is believed to have anti-carcinogenic effects, while the trans 10 cis 12 (T10C12) isomer is believed to be associated with anti-obesity effects. In this paper we extend earlier Molecular Dynamics (MD) simulations of pure CLA-phosphatidylcholine bilayers to investigate the comparative effects of cholesterol on bilayers composed of the two respective isomers. Simulations of phosphatidylcholine lipid bilayers in which the sn-2 chains contained one of the two isomers of CLA were performed in which, for each isomer, the simulated bilayers contained 10%, and 30% cholesterol (Chol). From MD trajectories we calculate and compare structural properties of the bilayers, including areas per molecule, thickness of bilayers, tilt angle of cholesterols, order parameter profiles, and one and two-dimensional radial distribution function (RDF), as functions of Chol concentration. While the structural effect of cholesterol is approximately the same for both isomers, we find differences at an atomistic level in order parameter profiles and in two-dimensional radial distribution functions.

Bis(monoacylglycero)phosphate (BMP) is an endosomal lipid with a unique structure that is implicated in the formation of intraendosomal vesicular bodies. Here we have characterized the effects of dioleoyl BMP (BMP18:1) at concentrations of 5, 10, 15 and 20 mol% on the thermotropic behavior of dipalmitoyl phosphatidylcholine (DPPC) vesicles, and compared them to those of equimolar concentrations of dioleoyl phosphatidylglycerol (DOPG), a structural isoform of BMP18:1. Because BMP is found in the acidic environments of the late endosome and intralysosomal vesicles, samples were prepared at pH 4.2 to mimic the pH of the lysosome. Both 2H NMR of perdeuterated DPPC and spin-labeled EPR with 16-Doxyl phosphatidylcholine were utilized in these investigations. NMR and EPR results show that BMP18:1 induces a lowering in the main phase transition temperature of DPPC similar to that of DOPG. The EPR studies reveal that BMP18:1 induced more disorder in the Lβ phase when compared to equimolar concentrations of DOPG. Analysis from dePaked 2H NMR spectra in the Lα phase reveal that BMP18:1 induces less disorder than equal concentrations of DOPG. Additionally, the results demonstrate that BMP mixes with other phospholipids as a phospholipid and not as a detergent molecule as once speculated.

It is now well established that dietary lipids are incorporated into macrophage and T-cell membrane microdomains, altering their structure and function. Within cell membranes, there are specific detergent-resistant domains in which key signal transduction proteins are localized. These regions are classified as “lipid rafts”. Rafts are composed mostly of cholesterol and sphingolipids and therefore do not integrate well into the fluid phospholipid bilayers causing them to form microdomains. Upon cell activation, rafts compartmentalize signal-transducing molecules, thus providing an environment conducive to signal transduction. In this review, we discuss recent novel data describing the effects of n-3 PUFA on alterations in the activation and functions of macrophages and T-cells. We believe that the modifications in these two disparate immune cell types are linked by fundamentally similar changes in membrane lipid composition and transmembrane signaling functions. We conclude that the outcomes of n-3 PUFA-mediated immune cell alterations may be beneficial (e.g., anti-inflammatory) or detrimental (e.g., loss of microbial immunity) depending upon the cell type interrogated.

Conjugated linoleic acids (CLA) are found naturally in dairy products. Two isomers of CLA, that differ only in the location of cis and trans double bonds, are found to have distinct and different biological effects. The cis 9 trans 11 (C9T11) isomer is attributed to have the anti-carcinogenic effects, while the trans 10 cis 12 (T10C12) isomer is believed to be responsible for the anti-obesity effects. Since dietary CLA are incorporated into membrane phospholipids, we have used Molecular Dynamics (MD) simulations to investigate the comparative effects of the two isomers on lipid bilayer structure. Specifically, simulations of phosphatidylcholine lipid bilayers in which the sn-2 chains contained one of the two isomers of CLA were performed. Force field parameters for the torsional potential of double bonds were obtained from ab initio calculations. From the MD trajectories we calculated and compared structural properties of the two lipid bilayers, including areas per molecule, density profiles, thickness of bilayers, tilt angle of tail chains, order parameters profiles, radial distribution function (RDF) and lateral pressure profiles. The main differences found between bilayers of the two CLA isomers, are (1) the order parameter profile for C9T11 has a dip in the middle of sn-2 chain while the profile for T10C12 has a deeper dip close to terminal of sn-2 chain, and (2) the lateral pressure profiles show differences between the two isomers. Our simulation results reveal localized physical structural differences between bilayers of the two CLA isomers that may contribute to different biological effects through differential interactions with membrane proteins or cholesterol.

The first total synthesis for the (Z)-16-methyl-11-heptadecenoic acid, a novel fatty acid from the sponge Dragmaxia undata, was accomplished in seven steps and in a 44% overall yield. The use of (trimethylsilyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to the long-chain protected 10-bromo-1-decanol followed by a second acetylide coupling to the short-chain 1-bromo-4-methylpentane, which resulted in higher yields. Complete spectral data is also presented for the first time for this recently discovered fatty acid and the cis double bond stereochemistry of the natural acid was established. The title compound displayed antiprotozoal activity against Leishmania donovani (IC50 = 165.5 ± 23.4 µM) and inhibited the leishmania DNA topoisomerase IB enzyme (LdTopIB) with an IC50 = 62.3 ± 0.7 µM.

Both L-α-lysophosphatidylinositol (LPI) and 2-arachidonoyl-sn-glycero-3-phosphoinositol (2-AGPI) have been reported to activate the putative cannabinoid receptor, GPR55. Recent microsecond time-scale molecular dynamics (MD) simulations and isothiocyanate covalent labeling studies have suggested that a transmembrane helix 6/7 (TMH6/7) lipid pathway for ligand entry may be necessary for interaction with cannabinoid receptors. Because LPI and 2-AGPI are lipid-derived ligands, conformations that each assumes in the lipid bilayer are therefore likely important for their interaction with GPR55. We report here the results of 70 ns NAMD molecular dynamics (MD) simulations of LPI and of 2-AGPI in a fully hydrated bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). These simulations are compared with a 70ns simulation of the cannabinoid CB1 receptor endogenous ligand, N-arachidonoylethanolamine (anandamide, AEA) in a POPC bilayer. These simulations revealed that (1) LPI and 2-AGPI sit much higher in the bilayer than AEA, with inositol headgroups that can at times be solvated completely by water; (2) the behavior of the acyl chains of AEA and 2-AGPI are similar in their flexibilities in the bilayer, while the acyl chain of LPI has reduced flexibility; and (3) both 2-AGPI and LPI can adopt a tilted headgroup orientation by hydrogen bonding to the phospholipid phosphate/glycerol groups or via intramolecular hydrogen bonding. This tilted head group conformation (which represents over 40% of the conformer population of LPI (42.2±3.3%) and 2-AGPI(43.7±1.4%) may provide a low enough profile in the lipid bilayer for LPI and 2-AGPI to enter GPR55 via the putative TMH6/7 entry port.

The physicochemical properties of a novel series of cholesterol-based cationic lipids in the presence of DOPE were studied by various techniques in an effort to correlate cationic lipid structure with transfection efficacy. It was found that while DOPE improves the β-gal activity of the active AC and MC derivatives, the overall zeta potential of the particles, pDNA complexation and condensation is not improved. This is in stark contrast with the tertiary amine derivative DC whose dispersion properties were improved and its monolayer surface potential is restored at high molecular surface density in the presence of DOPE. Overall the transfection activity mediated by DC and the quaternary ammonium TC derivative was greatly improved in the presence of DOPE and is attributed to decreased cytotoxicity, improved fusogenicity and cellular association.

Deuteration at C-4 and C-5 of sphingosine was achieved via a hydrogen–deuterium exchange reaction of a β-ketophosphonate intermediate catalyzed by ND4Cl in D2O/tetrahydrofuran. To install deuterium at C-3 of sphingosine and sphingomyelin, sodium borodeuteride reduction/cerium(III) chloride reduction of an α,β-enone in perdeuteromethanol was used.

Highlights

► Outlined the need of novel strategies for cancer therapies that can counteract problems arising particularly in chemotherapy due to resistance to current drugs and their low specificity. ► Elaborated the differences in membrane composition and properties between cancer and non-cancer cells, the basis for the use of anticancer peptides derived from host defense peptides as new weapons against cancer. ► Described the current knowledge on the mode of action of these peptides and the status of in vivo studies. ► Summarized the challenges and perspectives for the development of host defense peptides as novel anticancer agents.

Although much progress has been achieved in the development of cancer therapies in recent decades, problems continue to arise particularly with respect to chemotherapy due to resistance to and low specificity of currently available drugs. Host defense peptides as effector molecules of innate immunity represent a novel strategy for the development of alternative anticancer drug molecules. These cationic amphipathic peptides are able to discriminate between neoplastic and non-neoplastic cells interacting specifically with negatively charged membrane components such as phosphatidylserine (PS), sialic acid or heparan sulfate, which differ between cancer and non-cancer cells. Furthermore, an increased number of microvilli has been found on cancer cells leading to an increase in cell surface area, which may in turn enhance their susceptibility to anticancer peptides. Thus, part of this review will be devoted to the differences in membrane composition of non-cancer and cancer cells with a focus on the exposure of PS on the outer membrane. Normally, surface exposed PS triggers apoptosis, which can however be circumvented by cancer cells by various means.

Host defense peptides, which selectively target differences between cancer and non-cancer cell membranes, have excellent tumor tissue penetration and can thus reach the site of both primary tumor and distant metastasis. Since these molecules kill their target cells rapidly and mainly by perturbing the integrity of the plasma membrane, resistance is less likely to occur. Hence, a chapter will also describe studies related to the molecular mechanisms of membrane damage as well as alternative non-membrane related mechanisms. In vivo studies have demonstrated that host defense peptides display anticancer activity against a number of cancers such as e.g. leukemia, prostate, ascite and ovarian tumors, yet so far none of these peptides has made it on the market. Nevertheless, optimization of host defense peptides using various strategies to enhance further selectivity and serum stability is expected to yield novel anticancer drugs with improved properties in respect of cancer cell toxicity as well as reduced development of drug resistance.

The conformations of model transmembrane peptides are studied to understand the structural and dynamical aspects of tetrameric bundles using a series of coarse grain (CG) molecular dynamics (MD) simulations since membrane proteins play a crucial role in cell function. In this work, two different amphipathic models have been constructed using similar hydrophobic/hydrophilic characteristics with two structurally distinct morphologies to evaluate the effect of roughness and hydrophilic topology on the structure of tetrameric bundles, one class that forms an ion-channel and one class that does not. Free energy calculations of typical amphipathic peptide topologies show that using a relatively smooth surface morphology allows for a stable conformation of the tetramer bundle in a diamond formation. However, the model with side chains attached to the core in order to roughen the surface has a stable square tetramer bundle which is consistent with experimental data and all-atom (AA) MD simulations. Comparisons of the CG simulations with AA MD simulations are in reasonable agreement with the formation of tetrameric homo-oligomers, partitioning within the lipid bilayer and tilt angle with respect to the bilayer normal. We concluded that a square or diamond shape tetrameric homo-oligomers could be stabilized by rational design of the peptide morphology and topology of the surface, thus allowing us to tune the permeability of the bundle or channel.

Diphytanoylphosphatidylcholine (DPhyPC) is a branched chain lipid often used for model membrane studies, including peptide/lipid interactions, ion channels and lipid rafts. This work reports results of volume measurements, water permeability measurements Pf, X-ray scattering from oriented samples, and x-ray and neutron scattering from unilamellar vesicles at T=30 °C. We measured the volume/lipid VL = 1426 ± 1 Å3. The area/lipid was found to be 80.5 ± 1.5 Å2 when both x-ray and neutron data were combined with the SDP model analysis (Kučerka et al., 2008); this is substantially larger than the area of DOPC which has the largest area of the common linear chain lipids. Pf was measured to be 7.0 ± 1.0 ×10−3 cm/sec; this is considerably smaller than predicted by the recently proposed 3-slab model (Nagle et al., 2008). This disagreement can be understood if there is a diminished diffusion coefficient in the hydrocarbon core of DPhyPC and that is supported by previous molecular dynamics simulations (Shinoda et al., 2004). While the DPhyPC head-head thickness (DHH= 36.4 Å), and Hamaker parameter (H=4.5 ×10−21J) were similar to the linear chain lipid DOPC, the bending modulus (KC=5.2 ± 0.5 ×10−21J) was 30% smaller. Our results suggest that, from the biophysical perspective, DPhyPC belongs to a different family of lipids than phosphatidylcholines that have linear chain hydrocarbon chains.

The chain length dependence of the interaction of PFOA, a persistent environmental contaminant, with dimyristoyl- (DMPC), dipalmitoyl- (DPPC) and distearoylphosphatidylcholine (DSPC) was investigated using steady-state fluorescence anisotropy spectroscopy, differential scanning calorimetry (DSC) and dynamic light scattering (DLS). PFOA caused a linear depression of the main phase transition temperature Tm while increasing the width of the phase transition of all three phosphatidylcholines. Although PFOA’s effect on the on Tm and the transition width decreased in the order DMPC > DPPC > DSPC, its relative effect on the phase behavior was largely independent of the phosphatidylcholine. PFOA caused swelling of DMPC but not DPPC and DSPC liposomes at 37°C in the DLS experiments, which suggests that PFOA partitions more readily into bilayers in the fluid phase. These findings suggest that PFOA’s effect on the phase behavior of phosphatidylcholines depends on the cooperativity and state (i.e., gel versus liquid phase) of the membrane. DLS experiments are also consistent with partial liposome solubilization at PFOA/lipid molar ratios > 1, which suggests the formation of mixed PFOA-lipid micelles.

2-O-Arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine was synthesized with carbon-13 enrichment of the three glycerol carbons and the carbonyl of the stearoyl group. Phospholipase A2 was utilized to give optically pure lyso-PC, and only 3% acyl migration occurred during reacylation with arachidonic acid anhydride. This phospholipid is an important biosynthetic precursor of arachidonic acid metabolites as well as the endocannabinoid 2-arachidonoylglycerol (2-AG), and is now available for NMR studies.

A series of new phosphatidylcholine analogues with structurally modified sn-2-substituents have been prepared. The synthetic compounds include oligo(ethyleneglycol) derivatives with chain-terminal pharmacophores that upon catalytic hydrolysis by phospholipaseA2 yielded a series of oligo(ethyleneglycol)-conjugates of the respective drugs. The approach here outlined may open a new way to employ OEG derivatives of phospholipids for therapeutic applications as secretory PLA2-targeted precursors of prodrugs.

Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid.

The first total synthesis for the (Z)-17-methyl-13-octadecenoic acid was accomplished in seven steps and in a 45% overall yield. The use of (trimethylsilyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to the long-chain protected 12-bromo-1-dodecanol followed by a second acetylide coupling to the short-chain 3-methyl-1-bromobutane, which resulted in higher yields. Complete spectral data is also presented for the first time for this recently discovered fatty acid. The title compound displayed antiprotozoal activity against Leishmania donovani (EC50 = 19.8 μg/ml) and inhibited the leishmania DNA topoisomerase IB at concentrations of 50 μM.

Cholesterol and selected derivatives were studied as mixed Langmuir monolayers with egg phosphatidylcholine (PC). As an extension of our earlier work, which employed binary sterol/PC mixtures, here we examined ternary mixed monolayers containing cholesterol along with an alternate sterol and PC in different molar ratios, using pressure-area isotherms. The ternary systems behaved similarly to the binary sterol/PC systems reported previously, with similar condensation noted for the sterol/PC films. To better understand how variations in sterol structure affect sterol packing in such membrane monolayers, binary mixtures containing cholestenone, cholestanol, and lanosterol with PC were also studied. Cholestanol behaved similarly to cholesterol when incorporated with PC, while cholestenone and lanosterol did not cause as much film condensation. The observed differences in molecular packing, and attributed sterol structural differences, are considered within the context of sterol/phospholipid mixtures in biological membranes.

X-band and Q-band electron paramagnetic resonance (EPR) spectroscopic techniques were used to investigate the structure and dynamics of cholesterol containing phospholipid bicelles based upon molecular order parameters (Smol), orientational dependent hyperfine splittings and line shape analysis of the corresponding EPR spectra. The nitroxide spin label 3-β-doxyl-5-α-cholestane (cholestane) was incorporated into DMPC/DHPC bicelles to report the alignment of bicelles in the static magnetic field. The influence of cholesterol on aligned phospholipid bicelles in terms of ordering, the ease of alignment, phase transition temperature have been studied comparatively at X-band and Q-band. At a magnetic field of 1.25 T (Q-band), bicelles with 20 mol% cholesterol aligned at a much lower temperature (313 K), when compared to 318 K at a 0.35 T field strength for X-band, showed better hyperfine splitting values (18.29 G at X-band vs. 18.55 G at Q-band for perpendicular alignment and 8.25 G at X-band vs. 7.83 G at Q-band for the parallel alignment at 318 K) and have greater molecular order parameters (0.76 at X-band vs. 0.86 at Q-band at 318 K). Increasing cholesterol content increased the bicelle ordering, the bicelle-alignment temperature and the gel to liquid crystalline phase transition temperature. We observed that Q-band is more effective than X-band for studying aligned bicelles, because it yielded a higher ordered bicelle system for EPR spectroscopic studies.

Serum opacity factor from Streptococcus pyogenes transfers the cholesteryl esters (CE) of ~100,000 plasma high density lipoprotein particles (HDL) to a CE-rich microemulsion (CERM) while forming neo HDL, a cholesterol-poor HDL-like particle. HDL, neo HDL, and CERM are distinct. Neo HDL is lower in free cholesterol and has lower surface and total microviscosities than HDL; the surface polarity of neo HDL and HDL are similar. CERM is much larger than HDL and richer in cholesterol and CE. Although the surface microviscosity of HDL is higher than that of CERM, they have similar total microviscosities because cholesterol partitions into the neutral lipid core. Because of its unique surface properties apo E preferentially associates with the CERM. In contrast, the composition and properties of neo HDL make it a potential acceptor of cellular cholesterol and its esterification. Thus, neo HDL and CERM are possible vehicles for improving cholesterol transport to the liver.